Several chemical analyses are done by automatic analyzing equipment in hospitals and laboratories. The level of automation varies, but present trend is to streamline the analyzing work as much as possible. As many more complicated analyses must be performed manually by highly trained laboratory personnel, the productivity and throughput of such laboratory testing services has been low. By increasing the automation capabilities of the laboratory, more of the work can be performed inside the automatic systems without involvement of highly skilled personnel. Ideally laboratory assistants simply load samples in racks into the analyzing apparatuses freeing laboratory chemists and biologists to focus on interpreting the results and managing the operation of the laboratory. Such a system provides good throughput combined with high certainty and quality of analytical results.
Automated instruments for various kind of chemical analysis have been widely used for decades. They may combine both routine and sophisticated assay techniques such as spectrophotometry, fluorometry, time resolved fluorometry, chromatographic methods etc. with automated sample dispensing from original sample containers. Automation is often required as test workload increases within healthcare and clinical laboratories, public or commercial research and service institutes, or industrial process control.
Automatic discrete analysis techniques use computer controlled automation to perform steps similar to those of manual methods. For example, a computer controlled robotic arm may be used to position a probe of a pipette to aspirate or deposit a fluid sample, buffer or reagent into or out of any of a plurality of sample receptacles, a plurality of reagent receptacles and a plurality of reaction cuvettes. This is a “discrete” analysis technique because each sample is deposited in a discrete reaction cuvette, which is then subjected to an assaying device such as colorimetric photometer, or the like. Typically several discrete analyses may be done on subsamples divided from a main sample placed on a receptacle. For example, from a serum sample, a panel of serum lipids (triglycerides, cholesterol and HDL-cholesterol) can be measured.
Many sample types, particularly human samples for clinical testing, are not ready for assay as collected and, instead, require a pretreatment step. For example, plasma or serum must be separated from whole blood; whole blood must be hemolyzed to release intracellular components; fecal, sputum or solid tissue samples must be homogenized and suspended in liquids. In many cases of human excretions or other biological and industrial materials, interfering proteins must be precipitated, or analytes extracted from the original sample matrix. So while automatic analyzers have significantly improved throughput rate of analytical testing service, manual sample pretreatment has become a primary bottleneck in daily work flow.
Manual processes not only add to the time and labor required to produce results but also increase the risk of errors. Errors during the analytical process have been addressed primarily by automation as automation standardizes pipetting and dispensing steps, and eliminates variation in timing and differences between individual technicians. Sample pretreatment, however, remains prone to problems and would benefit from automation as well.
To increase sample throughput and provide process standardization, some automated or semi-automated sample pretreatment instruments have been developed. These are mainly meant to be used in connection with highly sophisticated analysis techniques that require sample purification steps before the actual measurement can be performed. For example, TurboFlow™ columns (Thermo Fisher Scientific) are used to separate small molecules from proteins or other large molecules in a sample before introduction to a LC/MS system. For PCR techniques, desired cell types must often be selected from complex sample mixtures for further treatment, and sample DNA or RNA must be purified to substantially eliminate background contamination before the actual cycling process. Semi-automated pretreatment instruments like those based on magnetic particles, e.g. the KingFisher™ instrument (Thermo Fisher Scientific), are useful for such processes.
Publications WO 2004096443, US 2005069913, U.S. Pat. Nos. 5,985,153 and 7,897,337 disclose examples of automated pretreatment methods and apparatuses and fully automated analyzing processes including pretreatment of all input samples. These are mainly used for DNA and RNA analysis wherein pretreatment is needed for all samples before analysis can be performed. Contamination risk is also extremely high. Usually disposable sample containers are needed, as well as disposable pipette tips or dispensers for transporting samples, sample aliquots, analytes and reagents.
However, many laboratories must function without such sophisticated automated or semi-automated equipment. Typically, the main daily workload of many clinical laboratories is performed using low-cost automated analyzers based on simple analytical techniques such as photometry or spectrophotometry. Such analyzers usually employ conventional assay methods that provide result levels traditionally considered sufficient for measurement of clinically significant ranges. Many compromises concerning acquired information and accuracy are made in order to achieve acceptable cost and speed while providing ease of use and maintenance.
Sample pretreatment is often considered as a separate process from the actual assay and, therefore, not well suited to instruments streamlined to perform one type of analytical process or measurement quickly and efficiently. Pretreatments are designed according to sample matrix and nature of analyte, and are usually not bound to particular assay principles. This may explain the lack of pretreatment automation, as sample oriented additional steps preceding the chemical assay reaction would lower throughput and cause difficulty in timing of assay sequences.
Despite difficulties in combining pretreatment processes with automatic discrete analysis, some pretreatment systems have been combined to automatic analysis apparatuses. Some of these systems are described below for reference.
On-Board Pretreatment of Whole Blood in Discrete Photometric Analyzers in the Measurement of Glycated Hemoglobin A1c (HbA1c).
In on-board methods to measure HbA1c, a sample of whole blood is hemolyzed within a discrete photometric analyzer to release the hemoglobin molecules from within blood cells. Hemolyzation is carried out by mixing whole blood with hemolyzing reagent in a reaction vessel or a cuvette and incubating the mixture for a defined time. After hemolyzation, an aliquot of the mixture is sampled to measure HbA1c content with a turbidimetric inhibition immunoassay and another aliquot is sampled to measure total hemoglobin content. In such method, the analytes and matrix components are not separated prior to the analyzing. This kind of analysis can be performed by Konelab™ clinical chemistry analyzers (Thermo Fisher Scientific), for example.
Cadmium Column Reduction of Nitrate
In a cadmium column, nitrate (NO3−) is reduced to nitrite (NO2−) using Cd2+ granules as catalyst. The reduction is accomplished by aspirating sample to the column where the reduction takes place and then eluting the reduced sample to a vessel. From the vessel an aliquot is sampled to measure nitrite. This kind of analysis can be performed by an Aquakem™ analyzer (Thermo Fisher Scientific). In this system analytes undergo a chemical reaction during the pretreatment; there is no means for separating reacted from unreacted or other non-target analytes. The nitrate reduction to nitrite can also be performed in a reaction vessel.
US 2009/0162942 discloses an automated discrete fluid sample analyzer that includes a sample preparation module. The sample preparation module includes a well configured to receive a sample deposited by a pipette and a sample preparation device in fluid communication with the well. The fluid sample is transferred from the well to the sample preparation device which prepares the sample by using catalyst, ultraviolet light or heat. The target analyte is not separated from the sample.
Samples of whole blood may have to be pretreated also in order to release a target analyte from components within the sample that bind or sequester the analyte. This can be done by preparation of a secondary specimen i.e. blood plasma or serum by centrifugation or filtration. A hemolysis reagent like saponin or detergent may be added followed by centrifugation or filtration. Likewise a denaturing agent may be used, e.g. acid (HCl, TCA) or an organic solvent (methanol, acetonitrile) or a combination of methanol and ZnSO, followed by centrifugation. These protocols involve manual steps which are difficult to automate or integrate into an on-line analysis procedure. In order to increase the degree of automation, an automated on-line hemolysis processor referred to as a Bloodlyser® device has been described (Morello, R. et al. Ther Drug Monit 29:143, 2007). As used, a sedimented blood sample is mixed by an autosampler procedure And the homogenized sample is pumped through a stainless steel capillary heated to 75° C. and the resulting heat causes no precipitation of blood/plasma proteins yet results in complete disintegration of blood cells. This leads to generation of new matrix referred to as “cell-disintegrated blood” by the developers of the process.